Peripheral artery disease (PAD) is increasingly prevalent and leads to disability and high cardiovascular risk. The determinants of clinical status and prognosis in PAD are not well understood. Emerging evidence from experimental models identifies microRNAs (miRNAs) as regulators of cellular responses to limb ischemia and as potential therapeutic targets. Altered miRNA expression may contribute to PAD by modulating angiogenesis, inflammation and endothelial injury response, but little is known about these mechanisms in patients. MiRNAs circulate in the blood, hold potential as biomarkers, and may function as endocrine signaling factors. Endothelial cells are a potential source for circulating miRNA. We have developed methods to perform comprehensive quantitative RT-PCR based miRNA profiling in plasma. In addition, we have established the ability to characterize miRNA expression in freshly isolated endothelial cells. The proposed research aims to define circulating and arterial endothelial miRNAs in PAD to provide insight into the mechanisms driving clinical disease in PAD. We will accomplish Aims 1 and 2 by studying our well-characterized, National Heart Lung and Blood Institute-funded PAD cohort and controls (306 PAD patients, 50 CAD patients and 50 controls) with longitudinal outcome evaluation and Aim 3 by enrolling 90 patients (30 each PAD, CAD alone and control) for endothelial cell isolation. In Aim 1, we will test the hypothesis that PAD alters circulating miRNA expression by comparing PAD patients to CAD and healthy controls. In Aim 2, we will relate circulating miRNAs to disease severity, inflammatory cytokines, and circulating angiogenesis modulators in PAD. Further, we will use previously collected 2-year follow-up data to test the hypothesis that circulating miRNAs are associated with coronary and peripheral disease events in PAD. In Aim 3, we will collect arterial endothelial cells at the time of angiography by J-wire biopsy from the iliofemoral arteries. We relate endothelial cell miRNA expression to circulating miRNAs and PAD status. These studies will provide new information regarding circulating and endothelial miRNAs in PAD and may stimulate innovative treatment strategies.